CN111781348A - Fluorescent quantitative immunochromatography detection test paper for pesticide ethofenprox and application thereof - Google Patents
Fluorescent quantitative immunochromatography detection test paper for pesticide ethofenprox and application thereof Download PDFInfo
- Publication number
- CN111781348A CN111781348A CN202010650202.6A CN202010650202A CN111781348A CN 111781348 A CN111781348 A CN 111781348A CN 202010650202 A CN202010650202 A CN 202010650202A CN 111781348 A CN111781348 A CN 111781348A
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- ethofenprox
- antibody
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- detection
- carrier protein
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Links
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses pesticide ethofenprox fluorescence quantitative immunochromatography detection test paper and application thereof. The invention provides a fluorescent quantitative immunochromatographic test strip for pesticide ethofenprox, which consists of a sample pad, a marker combination pad (provided with an anti-ethofenprox antibody marked by fluorescent microspheres), a chromatographic membrane and a water absorption pad, wherein the sample pad, the marker combination pad, the chromatographic membrane and the water absorption pad are sequentially connected and fixed on a bottom plate, the chromatographic membrane is provided with a detection line (close to the sample pad, coated with a conjugate of phenylacetic acid-ethofenprox azo compound and carrier protein 1) and a quality control line (close to the water absorption pad, coated with an antibody for resisting the ethofenprox antibody marked by the fluorescent. By using the inventionThe test paper provided for detecting the pesticide ethofenprox has the advantages of low detection limit, high accuracy, strong specificity, good precision and standard curve equation R2The value can reach 0.9801, and the detection result can be obtained within 10 min. Therefore, the ethofenprox fluorescence quantitative immunochromatographic test paper card prepared by the invention is suitable for rapid and quantitative detection of ethofenprox pesticide.
Description
Technical Field
The invention relates to the technical field of biology, in particular to fluorescent quantitative immunochromatography detection test paper for pesticide ethofenprox and application thereof.
Background
Pesticide residues are becoming more and more a concern in the industries of agriculture and medicinal plant production, food and drug safety, import and export and the like. Particularly, in the export trade of traditional Chinese medicinal materials, the over-high pesticide residue often becomes a 'neck clamp' factor, and great loss is caused to China. Recently, the requirements of the national departments on pesticide residue are becoming more and more strict, and thus the detection of pesticide residue is receiving more and more attention.
Currently, the common methods for detecting pesticide residues are mainly divided into two categories: one is analysis by means of large or precise instruments, such as High Performance Liquid Chromatography (HPLC), high performance liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), etc.; the other is a rapid and simple method, such as dry chemical method, enzyme linked immunosorbent assay (ELISA), colloidal gold immunochromatography, and the like. The first method has the advantages of high detection precision and guaranteed accuracy of quantitative analysis; the defects mainly include high requirements on knowledge and technology of operators, expensive detecting instruments, complex using process, long detecting time, unsuitability for field detection and the like. The second method has the advantages of simple operation, low requirement on operators, quick detection, low dependence on instruments and the like. The colloidal gold immunochromatography (gold-labeled test paper) is a representative detection means, has the advantages of being on-site, rapid, free of instruments and equipment, capable of directly judging results and the like, belongs to a quantitative or semi-quantitative method, and has certain difference in sensitivity compared with other methods.
The fluorescent microsphere immunochromatographic test paper made of the fluorescent microspheres instead of colloidal gold is combined with a portable immunochromatographic detector, so that the advantages of simple and convenient gold-labeled test paper, rapidness, field use and the like are kept, meanwhile, the accurate quantitative detection can be realized, and the sensitivity is greatly improved. The ethofenprox is a high-efficiency and low-toxicity pesticide, and has the characteristics of wide insecticidal spectrum, high knockdown speed, high insecticidal activity, long lasting period and the like, so that the ethofenprox is widely applied.
Disclosure of Invention
The invention aims to provide a fluorescent quantitative immunochromatography test paper for pesticide ethofenprox and application thereof.
In a first aspect, the invention claims a fluorescent quantitative immunochromatography test strip for pesticide ethofenprox.
The invention discloses a fluorescent quantitative immunochromatographic test strip for pesticide ethofenprox, which consists of a sample pad, a marker combination pad, a chromatographic membrane (such as an NC membrane) provided with a detection line and a quality control line and a water absorption pad which are sequentially connected and fixed on a bottom plate (such as a PVC material).
The label combining pad is provided with an anti-ethofenprox antibody marked by fluorescent microspheres.
The detection line and the quality control line are separated from each other.
The detection line is coated with a conjugate of phenylacetic acid-ethofenprox azoate (shown in formula I) and carrier protein 1.
And a secondary antibody is coated on the quality control line, and the secondary antibody is an antibody against the anti-ethofenprox antibody marked by the fluorescent microspheres.
The detection line is located at one end of the chromatographic membrane close to the sample pad.
The quality control line is positioned at one end of the chromatographic membrane close to the water absorption pad.
In the invention, the anti-ethofenprox antibody is a polyclonal antibody obtained after a rabbit is immunized by taking a conjugate of 4-oxo-4- (3-phenoxybenzyloxy) butyric acid (shown as a formula II) and carrier protein 2 as an immunogen.
The rabbit may be a big ear white rabbit.
In a particular embodiment of the invention, the carrier protein 1 is in particular chicken Ovalbumin (OVA) and the carrier protein 2 is in particular Bovine Serum Albumin (BSA).
In a specific embodiment of the invention, the coupling ratio of the phenylacetic acid-etofenprox azo compound to the egg white protein is 8: 1; the coupling ratio of the 4-oxo-4- (3-phenoxybenzyloxy) butyric acid to the bovine serum albumin is 15: 1. The ratio may also vary from batch to batch in actual studies. Different coupling ratios of the antigens and reagent ratios of the final test strip may also vary accordingly. As long as the use is not affected.
The conjugate of the phenylacetic acid-etofenprox azo compound and the carrier protein 1 can be prepared by a method comprising the following steps: preparing active ester of the phenylacetic acid-etofenprox azo compound, then adding the carrier protein 1, and reacting to obtain the conjugate of the phenylacetic acid-etofenprox azo compound and the carrier protein 1.
The conjugate of the 4-oxo-4- (3-phenoxybenzyloxy) butyric acid and the carrier protein 2 can be prepared by a method comprising the following steps: preparing the active ester of the 4-oxo-4- (3-phenoxybenzyloxy) butyric acid, then adding the carrier protein 2, and reacting to obtain the conjugate of the 4-oxo-4- (3-phenoxybenzyloxy) butyric acid and the carrier protein 2.
The anti-ethofenprox antibody marked by the fluorescent microsphere is a polymer formed by covalently combining the anti-ethofenprox antibody to be marked and the fluorescent microsphere through amido bond.
The diameter of the fluorescent microsphere is 330nm, the exciting light is 475nm, and the fluorescence is 525 nm.
The anti-ethofenprox antibody marked by the fluorescent microspheres can be prepared by the method comprising the following steps: diluting the fluorescent microspheres by using a dilution buffer solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxy thiosuccinimide, and incubating for 1h at 37 ℃; then adding the anti-ethofenprox antibody, and incubating for 1h at 37 ℃; then adding a sealing liquid for sealing (10 minutes); and centrifuging, and obtaining a precipitate containing the anti-ethofenprox antibody marked by the fluorescent microspheres.
In the present invention, in the preparation of the anti-ethofenprox antibody, the immunization procedure of rabbits (such as white rabbits with big ear) with the conjugate of 4-oxo-4- (3-phenoxybenzyloxy) butyric acid and carrier protein 2 as an immunogen is as follows:
first immunization: after mixing the 4-oxo-4- (3-phenoxybenzyloxy) butyric acid conjugate with the carrier protein 2 and Freund's complete adjuvant, the rabbits were immunized by multipoint intradermal injection, and the anti-ethofenprox antibody was prepared by using the 4-oxo-4- (3-phenoxybenzyloxy) butyric acid conjugate with the carrier protein 2 at an amount of 200-.
And (3) boosting immunity: two weeks after the first immunization, performing a first booster immunization, mixing the conjugate of 4-oxo-4- (3-phenoxybenzyloxy) butyric acid and carrier protein 2 with Freund's incomplete adjuvant, and injecting the mixture into immunized rabbits by multipoint intradermal injection, wherein the amount of the conjugate of 4-oxo-4- (3-phenoxybenzyloxy) butyric acid and carrier protein 2 is 50-400 μ g/rabbit (for example, 300 μ g/rabbit); two weeks after the first booster immunization, the second booster immunization was performed in the same manner and dose as the first booster immunization.
Further, in the preparation process of the anti-ethofenprox antibody, blood is collected 10 days after the second boosting immunization, and the prepared serum is the multi-antiserum; and then performing Protein G column purification on the multi-antiserum, dialyzing and concentrating PEG to obtain the anti-ethofenprox antibody.
In the purification of the Protein G column, the column was eluted with a glycine solution of pH2.5, and the resulting eluate was neutralized with a Tris buffer of 1M pH 9.0.
The dialysis is carried out by pure water.
In a particular embodiment of the invention, the secondary antibody is in particular goat anti-rabbit IgG.
Wherein the width of the label combining pad is 3.5mm, and the amount of the fluorescent microsphere labeled anti-ethofenprox antibody sprayed on the label combining pad is 4 mu g/cm2(calculated by the amount of the anti-ethofenprox antibody), the coating amount of the conjugate of the phenylacetic acid-ethofenprox azo compound and the carrier protein 1 on a detection line is 0.06 mu g/mm, and the coating amount of the secondary antibody on a quality control line is 0.06 mu g/mm; or the width of the label combining pad is 3mm, and the amount of the fluorescent microsphere labeled anti-ethofenprox antibody sprayed on the label combining pad is 4 mu g/cm2(calculated as the amount of the anti-ethofenprox antibody), the coating amount of the conjugate of the ethofenprox hapten and carrier protein on a detection line is 0.08 mu g/mm, and a quality control lineThe coating amount of the secondary antibody is 0.06. mu.g/mm.
In a second aspect, the invention claims a pesticide ethofenprox fluorescence quantitative immunochromatography detection test paper card.
The invention discloses a pesticide ethofenprox fluorescence quantitative immunochromatography detection test paper card, which consists of the test paper strip and a cover plate, wherein the cover plate is buckled with the test paper strip and meets the following conditions: the cover plate is provided with a sample adding window corresponding to the sample pad and a display window corresponding to the detection line and the quality control line.
In a third aspect, the invention claims any of the following:
(A1) the phenylacetic acid-ethofenprox azo compounds described hereinbefore;
(A2) a conjugate of the aforementioned phenylacetic acid-ethofenprox azo compound and carrier protein 1;
(A3) the anti-ethofenprox antibodies described previously;
(A4) the anti-ethofenprox antibody labeled with the fluorescent microsphere described above;
(A5) a composition consisting of the conjugate of 4-oxo-4- (3-phenoxybenzyloxy) butyric acid with carrier protein 2 and (a4) as described above.
In a fourth aspect, the invention claims any of the following applications:
(B1) the use of the test strip or card or substance as described above for the detection of ethofenprox;
(B2) use of a substance as hereinbefore described in the preparation of a test strip or card as hereinbefore described.
In a fifth aspect, the invention claims a method for detecting the content of ethofenprox in a sample to be detected.
The method for detecting the content of ethofenprox in the sample to be detected, which is claimed by the invention, can comprise the following steps:
(C1) drawing a standard curve: preparing standard substance solutions of etofenprox with a series of concentrations, respectively detecting by the test strip or the test paper card, adding the standard substance solutions into the sample pad of the test paper strip or the sample adding window of the test paper card, and reacting for 10 min; reading the fluorescence intensity of the quality control line and the detection line by using a fluorescence reader; drawing a standard curve graph by taking the concentration of the standard substance of the ethofenprox as an abscissa and the fluorescence intensity ratio of the detection line and the quality control line as an ordinate to obtain a standard curve equation;
(C2) detecting a sample to be detected: adding the sample to be detected into the sample pad of the test strip or the sample adding window of the test strip card, and reacting for 10 min; and (3) reading the fluorescence intensities of the quality control line and the detection line of the test strip by using a fluorescence reading instrument, substituting the fluorescence intensity ratio of the detection line and the quality control line into the standard curve equation obtained in the step (C1), and calculating to obtain the concentration value of ethofenprox in the sample to be detected.
The fluorescent microspheres used as the signal indicator and the portable detector can effectively overcome the defects of the colloidal gold immunochromatography and realize the real quantitative detection of the pesticide ethofenprox.
One of the factors determining the quality of the immunochromatographic test strip is the binding property of an antigen and an antibody. The antigen and the antibody used in the invention are prepared by self, and the reaction of the antigen and the antibody is measured to have high sensitivity and good specificity; and a solid substance foundation is laid for the development of the test paper.
The invention evaluates the performance of the prepared test paper in the aspects of detection limit, accuracy, specificity, precision and the like, and the experimental result shows that the lower detection limit of the ethofenprox test paper can reach 1.057ng/mL (card 1) and 1.289ng/mL (card 2); the addition recovery rate of the ethofenprox standard substance is close to 100 percent, which shows that the accuracy is very high. The cross reaction rate of bifenthrin and permethrin with similar pesticides of ethofenprox is lower than 0.10 percent, and the high specificity is shown. When the ethofenprox standard substance with series concentration is detected, the variation coefficient in batch and between batches is lower than 10.00 percent, and the requirement of the detection reagent on precision is completely met. In addition, the equation of the standard curve R2The values reached 0.9801 (card 1) and 0.9696 (card 2), and the detection results were obtained within 10 min; therefore, the ethofenprox fluorescence quantitative immunochromatographic test paper card prepared by the invention is suitable for rapid and quantitative detection of ethofenprox pesticide.
Drawings
FIG. 1 shows the structure of etofenprox (MW 376).
FIG. 2 shows the synthesis route of phenylacetic acid-etofenprox azo compound.
FIG. 3 is a scheme for the synthesis of 4-oxo-4- (3-phenoxybenzyloxy) butyric acid.
FIG. 4 is a mass spectrum (first order) of hapten M coupling solution-mass spectrometry.
FIG. 5 is the detection mass spectrum (first order) of hapten M D.
FIG. 6 shows the Maldi-TOF mass spectrum of complete antigen M-BSA
FIG. 7 is a Maldi-TOF mass spectrum of complete antigen M-OVA.
FIG. 8 is a complete antigen SDS-PAGE detection.
FIG. 9 is a purified polyclonal antibody SDS-PAGE assay. Lanes 1-4 are purified polyclonal antibodies from rabbit antisera 1-4, respectively. Wherein, rabbit No. 1, No. 2 is immunized by 4-oxo-4- (3-phenoxybenzyloxy) butyric acid and BSA conjugate; rabbits No. 3, 4 were immunized with phenylacetic acid-ethofenprox azo with OVA conjugate.
Figure 10 is a multiple-antibody competitive inhibition regression curve. Antibody 2 is polyclonal antibody purified from rabbit No. 2 (M-BSA immune) serum; antibody 3 is a polyclonal antibody purified from rabbit No. 3 (M-OVA immune) serum.
FIG. 11 is a schematic diagram of a test strip structure.
Fig. 12 is a schematic view of a test paper card structure.
FIG. 13 shows the test strip effectiveness test.
Fig. 14 is a card 1 standard curve.
Fig. 15 is a card 2 standard curve.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 Synthesis of pesticidal Ethothrin antigen and preparation of antibody
Materials and methods
1. Main reagent, material and experimental animal
Succinic anhydride, 3-benzyloxybenzyl alcohol, Bovine Serum Albumin (BSA), egg white albumin (OVA), N-hydroxysuccinimide (NHS), N-Dimethylformamide (DMF), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and p-aminophenylacetic acid were purchased from Sigma, USA; sodium nitrite, hydrochloric acid, sodium hydroxide were purchased from Beijing national drug group; freund's complete adjuvant, Freund's incomplete adjuvant were purchased from Gibco corporation; ethofenprox standard was purchased from Olympic Biotechnology, Inc. of Beijing century; tetramethylbenzidine (TMB), biotinylated goat anti-rabbit IgG, and avidin-modified HRP were purchased from dake biotechnology gmbh; protein G antibody purification pre-packed column (1mL) is GE product; the ELISA plate is a Coasta product; white big ear rabbit, male, 15 weeks old.
2. Main instrument
Microplate reader (Biotek, Synergy H1); ultramicro UV-visible spectrophotometers (Denovix, DS-11FX), high-speed cryogenic centrifuges (SIGMA, 4-16K), ultra pure water systems (Millipore, Milli-Q); an electric heating constant temperature water bath box (Shanghai Senxin, DK-600B); electromagnetic stirrers (Tester, SH-2), electrophoresis apparatus (Bio-Rad, Mini Protean), Maldi-TOF mass spectrometer (Illuminus TOF200), liquid-mass spectrometry system (angioent 6410Triple Quad).
3. Synthesis of ethofenprox artificial hapten
The structure (molecular weight 376) of ethofenprox is shown in FIG. 1.
(1) Synthesis of phenylacetic acid-etofenprox azo compound
The synthesis reaction of phenylacetic acid-etofenprox azo compound is divided into two steps, as shown in figure 2.
Firstly, 16mg of p-aminophenylacetic acid is dissolved in precooled 2N hydrochloric acid under the condition of ice-water bathAdding 0.5M NaNO while stirring2And monitoring by potassium iodide starch test paper, and stopping when the test paper turns blue. Then 25mg of ethofenprox standard substance is put into borate buffer solution with the pH value of 9.0; and (3) performing ice water bath, adding the prepared diazo p-aminophenylacetic acid salt into the ethofenprox suspension while stirring, and continuing stirring for 30min after the addition is finished. A small amount of HCl was added to the resulting red liquid, a red precipitate was formed, and the supernatant was discarded after centrifugation. The precipitate is phenylacetic acid-etofenprox azo compound (M couple for short, molecular weight prediction 538), and is dried for standby.
(2) Synthesis of an ethofenprox ester analogue, 4-oxo-4- (3-phenoxybenzyloxy) butyric acid
The synthetic route of 4-oxo-4- (3-phenoxybenzyloxy) butyric acid is shown in figure 3.
0.8mg of succinic anhydride and 29mg of 3-phenoxybenzyl alcohol were dissolved in 50mL of dichloromethane, and stirred at room temperature for 5 hours, and the resulting liquid was washed with water, dried over anhydrous sodium sulfate, and then placed in a fume hood for suction drying to obtain 4-oxo-4- (3-phenoxybenzyloxy) butyric acid as white crystals (abbreviated as M-butyl, predicted molecular weight 300).
4. Active ester method for preparing ethofenprox complete antigen
Respectively taking 0.5mmol of the two artificial haptens (M pair and M butyl) prepared in the step 3, dissolving the two artificial haptens in 0.5mL of DMF, adding equal mol of EDC and NHS while stirring, continuously stirring for 6h, and cooling at 4 ℃ for 3h to obtain respective active esters. Centrifuging to obtain supernatant, and standing at 4 deg.C. 6mg BSA and 8mg OVA were dissolved in 5mL pH9.0 carbonate buffer to obtain solutions B and O, respectively. And (3) electromagnetically stirring, namely dropping the M-coupled active ester into the O solution, dropping the M-butyl active ester into the B solution, and continuously stirring overnight after dropping. Centrifuging to obtain supernatant, placing in dialysis bag, dialyzing with 1L pure water at 4 deg.C, and changing the medium for 5 times. Concentrating with PEG (polyethylene glycol) to obtain complete antigen (named M-OVA and M-BSA), lyophilizing, and storing at-80 deg.C.
5. Synthetic antigen identification
(1) Artificial antigen LC-MS detection
Weighing 1mg of the two artificial haptens (M pair and M butyl) prepared in the step 3 respectively to ensure that the final concentration of the M butyl is 30 mu g/mL and the final concentration of the M pair is 10 mu g/mL, and the chromatographic conditions comprise a chromatographic column ZORBAXSB-C182.1 × 50mm and 1.8 mu M, and a mobile phase M butyl is 100% methanol, M-couple A phase is 0.1% aqueous ammonia solution, and B phase is methanol (% by volume). The sample injection amount is 1 mu L, and the M is even 5 mu L. Mass spectrum conditions: ESI ion source, M-positive ion mode, M-negative ion mode; atomization pressure 45PSI, drying gas (N)2) The flow rate is 10L/min, the temperature of the drying gas is 350 ℃, the capillary voltage M is 4000V, and M is 2500V; and (3) in a full scanning mode, wherein the scanning range is M/z100-400, and M even M/z 450-650, and a mass spectrogram (primary level) is obtained.
(2) Complete antigen detection
Preparing M-OVA, M-BSA and OVA and BSA aqueous solutions with the concentration of 0.5mg/mL, carrying out 2 mu L of dot target, carrying out acetonitrile matrix, and carrying out Maldi-TOF mass spectrometry to obtain an ion mass spectrogram. And (4) calculating the coupling ratio according to the molecular weight change before and after the coupling of the OVA and the BSA with the hapten in the map.
Two complete antigens were simultaneously detected by SDS-PAGE with OVA and BSA as controls.
6. Preparation of Ethothrin polyclonal antibody
(1) Preparation of multiple antisera
Selecting 4 male big-ear white rabbits with the size of 15 weeks as immune objects, and numbering No. 1-4; 5 mL/blood is collected from the marginal vein of the ear before immunization, serum is obtained by centrifugation, and the serum is used as negative control serum and is stored at the temperature of minus 20 ℃ for standby. Two complete antigens (M-OVA and M-BSA) (No. 1, No. 2 rabbit with M-BSA and No. 3, No. 4 rabbit with M-OVA) were dissolved in physiological saline at a concentration of 1 mg/mL; according to the immunization dosage, the antigen solution is mixed with Freund's adjuvant with the same volume, the mixture is made into water-in-oil emulsion by vortex oscillation, and the multipoint intradermal injection immunization is carried out. The dosage of the primary immune antigen is 400 mug/rabbit, and Freund's complete adjuvant is used; the immunization is strengthened once after two weeks, and the dosage of the antigen is 300 mug/rabbit; freund's incomplete adjuvant was used. 8 days after the second boosting immunization, 5 mL/rabbit of blood is collected from the ear marginal vein, serum is prepared, the complete antigen is used for coating, and ELISA is carried out to detect the serum titer; greater than 1: 128000, on day 10, a large number of blood samples were collected from the marginal artery to prepare serum, which was a polyclonal antiserum.
(2) Multi-antiserum purification
10mL of PB buffer solution with the pH value of 7.2 is sucked by the injector, and the equilibrium Protein G antibody purification pre-packed column is slowly washed; then, antiserum diluted 3 times with PB was slowly injected into the purification column, and the effluent liquid at the lower end of the column was allowed to flow out dropwise. Eluting with 5mL PB, eluting with 3mL of glycine solution with pH2.5, neutralizing the eluate with 1M Tris buffer solution with pH9.0, and dialyzing with pure water in dialysis bag; concentrating with PEG, detecting concentration with ultraviolet, and freezing at-80 deg.C.
7. Identification of Ethothrin polyclonal antibody
(1) Multi-antibody purification effect and potency determination
M-OVA and M-BSA were dissolved at 200ng/mL in pH9.0 carbonate buffer, and a 96-well ELISA plate was coated at 100. mu.L/well overnight at 4 ℃. After washing the plate 2 times, M-OVA coated wells were blocked with 1% (% expressed as g/100mL) OVA in 0.02M PBST, and M-BSA coated wells were blocked with 1% (% expressed as g/100mL) BSA in 0.02M PBST at 150. mu.L/well for 2h at 37 ℃. Washing the plate for 2 times, adding purified antibody diluted by washing liquor in a gradient manner into corresponding holes at a rate of 100 mu L/hole, wherein M-OVA immune antibody is added into M-BSA coated holes, M-BSA immune antibody is added into M-OVA coated holes, and washing liquor is added into negative control holes; incubate at 37 ℃ for 1.5 h. Washing the plate for 5 times, adding biotinylated goat anti-rabbit IgG-HRP into the plate at a concentration of 100 mu L/hole, and keeping the temperature at 37 ℃ for 1 h; after washing the plate for 5 times, adding avidin HRP at 100 mu L/hole, and keeping the temperature at 37 ℃ for 0.5 h; washing the plate for 5 times, and adding TMB color development liquid at 50 mu L/hole; developing at 37 deg.C for 20min, and adding stop solution; the absorbance values were read at 450nm with a microplate reader. And (4) detecting whether the OD value of the detection hole is more than 2 times of the OD value of the negative hole is positive, and determining the maximum dilution of the positive hole as the antibody titer. In addition, each polyclonal antibody was detected by SDS-PAGE electrophoresis.
(2) IC50 determination
According to the titer measurement experiment results, 1 antibody with high titer in 2 antigens was selected to detect the IC50 (50% inhibition concentration). The antibody dilution with an OD of about 1.5 was selected as the standard for IC50 detection. Gradient ethofenprox standard solutions (1, 10, 20, 30, 40, 50, 60, 70 and 80ng/mL) are prepared, 50 μ L of each standard solution is taken, 50 μ L of diluted antibody is added to each standard solution to serve as ELISA detection samples, and other operations are the same as the step (1).
Second, result in
1. Artificial antigen identification
The two prepared artificial antigens M-OVA and M-BSA are respectively detected by LC-MS, and the obtained ion mass spectrogram is analyzed to obtain that M couple is mainly detected in the form of negative ion molecules combined with one molecule of boric acid molecules, and M couple is mainly detected in the form of positive ion molecules combined with one sodium atom. The synthetic antigen molecular weight was consistent with that expected, and the artificial antigen synthesis was successful (fig. 4, fig. 5).
2. Complete antigen identification
The complete antigen is detected by mass spectrometry, and the obtained map shows that the molecular weight of the complete antigen is obviously shifted (increased) to the right compared with that of the carrier protein, which indicates that the artificial antigen is successfully coupled to the carrier protein (figure 6 and figure 7). Protein electrophoresis also demonstrates this: the molecular weight of the complete antigen is larger than that of the carrier protein, the swimming speed is slow, and the band is close to the upper part (figure 8). From the data of the molecular weight of the complete antigen and the carrier protein obtained from the mass spectrum result, the coupling ratio of M-OVA can be calculated to be 8:1, and the coupling ratio of M-BSA is 15: 1.
3. Polyclonal antibody identification
(1) Purification effect of polyclonal antibody
Purified antibody SDS-PAGE gels show that: there is a clear band at about 53kD, which should be a heavy strand; there was a clear band at about 23kD, which should be a light strand; meanwhile, a diffuse band exists in a light chain region, which is caused by heterogeneity of a polyclonal antibody (the molecular weight of the antibody aiming at various epitopes is different to a certain extent), and a heavy chain is difficult to disperse and is in a shape of a single chain due to post-translational modification of a C-terminal. In addition, there were no additional bands, indicating that the purity of the polyclonal antibodies was higher (FIG. 9).
(2) Polyclonal antibody titer detection
ELISA detection results show that 4 purified polyclonal antibodies (four antibodies corresponding to four lanes in FIG. 9) have higher titer (Table 1); with the titer, the requirements of manufacturing the ethofenprox immunodetection reagent are completely met. In ELISA experiments, the coating antigens detected by the anti-M-OVA antibody and the anti-M-BSA antibody are M-BSA and M-OVA respectively; avoiding antigen-antibody reaction against carrier protein in polyclonal antibody; thus, the specificity of the antibody was confirmed to be excellent.
TABLE 1 purified antibody ELISA assay titers
Note: in the table, antibodies 1 to 4 are antibodies purified from rabbit sera # 1 to # 4, respectively.
4. Polyclonal antibody IC50 detection
The competitive ELISA test results were fitted to a curve from which the IC50 for 2 antibodies (antibody 2 and antibody 3 in table 1) were calculated as: 40.28ng/mL and 43.81 ng/mL. The obtained polyclonal antibody has good specificity and sensitivity (FIG. 10).
Example 2 preparation and application of pesticide ethofenprox fluorescence quantitative immunochromatography detection test paper
Materials and methods
1. Experimental Material
M-OVA, rabbit anti-M-BSA polyclonal antibody (denoted Ab-M, antibody 2 in Table 1 of example 1) prepared in example 1; etofenprox, bifenthrin and permethrin standards are purchased from Olympic biotechnology limited of Beijing century; other chemical reagents were purchased from Beijing Wangjia Biotech, Inc.; fluorescent microspheres (diameter 330nm, excitation light 475nm, fluorescence 525nm, oceaneno TECH, cat # 110315), Nitrocellulose (NC) membrane, sample pad (glass fiber material), marker pad (non-woven material), absorbent pad (paper), polyvinyl chloride (PVC) back plate, test paper card plastic shell were purchased from beijing jinyuan and biotechnology limited; the film spray machine (IsoFlow Reagent) and slitter (Guillotine Cutter) are available from ARISTA, USA, and the portable fluorescence immunochromatography analyzer (FIC-H1) is available from Hangzhou Peak navigation technology, Inc.
2. Principal solution
NC membrane coating buffer was 0.01M PBS (phosphate buffered saline, phosphate buffer), containing 1% (1g/100mL) trehalose. The microsphere-labeled antibody labeling buffer solution was prepared with 0.01M PBS (pH7.2), and added with 1% (1g/100mL) BSA, 1% (volume percentage) goat serum, 5% (5g/100mL) sucrose, 0.05% (0.05g/100mL) EDTANA2. The dilution buffer was 0.05M borate buffer (BBS, pH 5.0). The storage buffer was 0.01M PBS (pH 7.4) containing 1% (1g/100mL) of polyethylene glycolAlcohol 2000, 5% (5g/100mL) sucrose, 1% (1g/100mL) BSA. The blocking solution was 0.01M Tris (pH 8.0) containing 0.04% (0.04g/100mL) BSA. Ethofenprox standard solution: standards were prepared in PBS at serial concentrations as required.
3. Fluorescent microsphere labeled antibody
(1) 100 μ L of fluorescent microspheres were diluted 1-fold with dilution buffer.
(2) 76.68. mu.L of a 0.125mg/mL solution of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 17.36. mu.L of a 0.125mg/mL solution of NHSS (N-hydroxythiosuccinimide) were added to solution (1) and mixed well, and incubated at 37 ℃ for 1 h.
(3) The antibody (Ab-M) to be labeled is diluted to 1mg/ml with PBS, 40. mu.L of the diluted antibody is mixed with the solution (2), and the mixture is incubated at 37 ℃ for 1 h.
(4) Adding 10 μ L of the sealing solution in the solution (3), mixing, sealing for 10min, and centrifuging at 8000rpm for 10 min.
(5) The supernatant was discarded, and 100. mu.L of the storage buffer was added for reconstitution.
4. Preparation of test paper cards
(1) Spraying M-OVA and goat anti-rabbit IgG (both concentration is 1mg/mL) dissolved in coating buffer solution onto NC membrane by using a membrane spraying machine, and respectively serving as a detection line (T line) and a quality control line (C line) to adjust spraying speed so that the amount of M-OVA on the membrane is respectively 0.05, 0.06 and 0.08 muL/mm; the amount of goat anti-rabbit IgG on the membrane is 0.06 muL/mm; drying with a dryer for later use.
(2) The mixed solution of the fluorescent-labeled antibody prepared in step 3 was diluted to 10. mu.L/cm2The amount of the composition was uniformly sprayed on the marker-binding pad, dried by a dryer, and cut into a series of strips of width (4.00, 3.50, 3.00, 2.50, 2.00mm) for use.
(3) The sample pad, the marker combination pad, the NC membrane and the water absorption pad obtained in the steps are sequentially overlapped and adhered along the length direction (chromatography direction) of the PVC backboard to manufacture a large test paper board, and the long marker combination pad strips with different widths and the NC membrane coated with different amounts of reagents form a series of proportional combination large boards of fluorescence labeling antibodies and M-OVA amount on the NC membrane, so that reagent parameters of the test paper are optimized conveniently, and the optimal reagent structure process of the test paper is selected. Each large panel was cut into strips of 3mm wide by a slitter, as shown in FIG. 11. The test paper is put into the card shell to be made into a test paper card, which is convenient for the detection of an instrument, as shown in figure 12.
5. Establishment of detection method and determination of test strip structure
(1) Detection method
Placing the test paper card on an experiment table, taking 20 mu L of standard solution or a sample to be tested, dripping into the test paper card from a sample hole, and dripping 100 mu L of dilution buffer solution into the hole; and (3) inserting the card into a detection port of a portable fluorescence immunochromatography analyzer after 10min, and scanning to obtain fluorescence values of a T line and a C line. The effectiveness detection is carried out firstly, the C line has an obvious numerical value at the wavelength of emitted light, namely the card test is effective, and the experiment can be continued. The ratio of the fluorescence scan values of the T and C lines (T/C) was used as the basis for quantification.
(2) Standard curve creation and detection limit determination
Dropwise adding the etofenprox standard solution with the series of concentrations as a sample into a sample hole of a test paper card, and scanning and reading by using a portable detector in the step (1); the measurement of the standard solution was repeated 3 times at each concentration, and the average value of the ratio T/C of the respective T-line and C-line readings was taken as the measurement value of the sample. Taking the series of T/C values as dependent variables and the concentrations of the standard substances corresponding to the T/C values as independent variables to make a standard curve, fitting a corresponding equation, and taking R as2Values greater than 0.95 are a useful requirement.
Detecting 20 blank dilutions, and repeatedly measuring for 10 times, wherein the average value of concentration values obtained by the obtained 20T/C values on a standard curve is added with 3 times of standard deviation to be used as a detection Limit (LOD); the mean plus 10-fold standard deviation was taken as the limit of quantitation (LOQ).
(3) Determination of optimal Structure of test strip
Preparing test paper cards with different widths of marker pads and different combinations of coating antigen amounts as in the step 4 (3); making respective standard curve according to the steps 5 and 2, calculating detection limit, and comparing the standard curve equation R2And obtaining the optimal combination by the detection limit, and determining the reagent proportion of the final test strip, namely determining the optimal structural process of the test strip.
6. Determination of the Properties of test cards
(1) Specificity of
Adding bifenthrin and permethrin with a concentration of 10ng/mL into the dilution buffer solution, measuring the T/C value by using a test paper card, obtaining a corresponding concentration value from a standard curve, and performing 6 repeated detections on each sample. The cross-reactivity rate was calculated from the data according to the formula, with lower values indicating better specificity.
The cross-reaction rate (%) × 100% (concentration detected in test card/added concentration).
(2) Accuracy of
Preparing ethofenprox standard substance solutions with the concentrations of 2.00, 10.00, 50.00 and 100.00ng/mL respectively, repeatedly measuring for 5 times by using a test card, obtaining corresponding concentrations by the obtained T/C values through a standard curve, and calculating the addition recovery rate.
The recovery rate is (concentration by assay/concentration by addition) × 100%.
The closer the recovery is to 100%, the higher the accuracy is represented.
(3) Precision degree
The ethofenprox quality control products with different concentration intervals (2.00, 10.00 and 50.00ng/mL) are detected by using the same batch of test paper cards and 3 different batches of test cards respectively, each mass concentration is tested for 10 times, and the average Coefficient of Variation (CV) between batches is calculated to represent the precision between batches.
Second, results and analysis
1. Validation of test cards
And adding the ethofenprox standard substance solution into the test paper card, scanning the detection line T and the quality control line C area by using a portable immunochromatography detector, and observing the fluorescence intensity. As a result, the standard substances with several measured concentrations have fluorescence emission peaks and corresponding numerical values on the line C; the test strip is shown to be effective. Meanwhile, the fluorescence intensity of the standard substances with different concentrations is in negative correlation with the concentration of the standard substances on the T line, and the effectiveness of the test paper is further explained. See fig. 13.
2. Standard curve and limit of detection
The standard curve is drawn by detecting ethofenprox standard solution with the concentration of 0, 10, 30, 50, 80, 110, 140, 170 and 200ng/mL by using a test paper card. Results the label-binding pad was 3.5mm wide and was M-OVA coatedR of the curve equation at a quantity of 0.06. mu.L/mm (denoted as card 1), a label-binding pad width of 3mm and a coating quantity of 0.08. mu.L/mm (denoted as card 2)2A value greater than 0.95; the proportion of the 2 reagents meets the preparation requirement of the test paper, and is shown in figure 14 and figure 15.
3. Optimum reagent structure process of test paper
And (3) detecting 20 parts of the diluent by using 2 test papers selected in the step (2), and obtaining concentration measurement values in respective corresponding standard curves so as to obtain respective detection limits and quantitative limits, which are shown in a table 2. Thus comparing the two, the card 1 (the width of the marker pad is 3.50mm, and the coating amount of M-OVA is 0.06 muL/mm) is the optimal combination; and the standard is used as the structural process standard of the final test paper card reagent.
Table 2 concentrations of two cards measured repeatedly on 20 dilutions
Limit of detection (LOD) and limit of quantitation (LOQ) (n 20, ng/mL)
4. Specificity of
The bifenthrin and permethrin with the concentration of 10ng/mL are respectively detected by a test paper card, and the cross reaction rates of the bifenthrin and permethrin are respectively as follows: 0.05% (bifenthrin) and 0.07% (permethrin), both less than 0.10%; the test paper card has good specificity.
5. Accuracy of
As shown in Table 3, the addition recovery rate of ethofenprox standard substance detection at each concentration is between 98.60% and 106.50%, which shows that the accuracy of the test paper card is high.
TABLE 3 recovery rate of addition of etofenprox standard at different concentrations
6. Precision degree
The precision of the test card was characterized by the coefficient of variation (CV value), and the results of the coefficient of variation between batches are shown in tables 4 and 5. The CV value in the batch ranges from 3.86% to 7.76%; the CV value between batches measured by 3 batches of test paper cards is between 5.02% and 7.83%. Both CV values are less than 10.00%, which indicates higher precision of the test paper card.
TABLE 4 Intra-batch coefficient of variation
TABLE 5 coefficient of variation between batches
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. The pesticide ethofenprox fluorescence quantitative immunochromatography detection test strip consists of a sample pad, a marker combination pad, a chromatography membrane and a water absorption pad, wherein the sample pad, the marker combination pad, the chromatography membrane is provided with a detection line and a quality control line, and the water absorption pad are sequentially connected and fixed on a bottom plate;
the label combination pad is provided with an anti-ethofenprox antibody marked by fluorescent microspheres;
the detection line and the quality control line are separated from each other;
the detection line is coated with a conjugate of phenylacetic acid-ethofenprox azo compound and carrier protein 1;
a secondary antibody is coated at the quality control line, and the secondary antibody is an antibody against the anti-ethofenprox antibody marked by the fluorescent microspheres;
the detection line is positioned at one end of the chromatographic membrane close to the sample pad;
the quality control line is positioned at one end of the chromatographic membrane close to the water absorption pad.
2. The test strip of claim 1, wherein: the anti-ethofenprox antibody is a polyclonal antibody obtained after a rabbit is immunized by taking a conjugate of 4-oxo-4- (3-phenoxybenzyloxy) butyric acid and carrier protein 2 as an immunogen.
3. The test strip of claim 1 or 2, wherein: the carrier protein 1 is chicken ovalbumin, and the carrier protein 2 is bovine serum albumin.
4. The test strip of claim 3, wherein: the coupling ratio of the pyrethrum ether hapten to the egg albumin is 8: 1; the coupling ratio of the 4-oxo-4- (3-phenoxybenzyloxy) butyric acid to the bovine serum albumin is 15: 1.
5. The test strip of claim 3 or 4, wherein: in the preparation process of the anti-ethofenprox antibody, the immune program of the rabbit immunized by using the conjugate of the 4-oxo-4- (3-phenoxybenzyloxy) butyric acid and the carrier protein 2 as immunogen is as follows:
first immunization: mixing the conjugate of the 4-oxo-4- (3-phenoxybenzyloxy) butyric acid and the carrier protein 2 with Freund's complete adjuvant, and injecting the immunized rabbit into multiple spots intradermally, wherein the dosage of the conjugate of the 4-oxo-4- (3-phenoxybenzyloxy) butyric acid and the carrier protein 2 is 200 μ g-;
and (3) boosting immunity: two weeks after the first immunization, performing first boosting immunization, mixing the conjugate of the 4-oxo-4- (3-phenoxybenzyloxy) butyric acid and the carrier protein 2 with Freund's incomplete adjuvant, and injecting the immunized rabbit by multipoint intradermal injection, wherein the dosage of the conjugate of the 4-oxo-4- (3-phenoxybenzyloxy) butyric acid and the carrier protein 2 is 50-400 mu g/rabbit; two weeks after the first booster immunization, the second booster immunization was performed in the same manner and dose as the first booster immunization.
6. The test strip of claim 5, wherein: in the preparation process of the anti-ethofenprox antibody, blood is collected 10 days after the second boosting immunization, and the prepared serum is the multi-antiserum; and then performing protein G column purification, dialysis and PEG concentration on the multi-antiserum to obtain the anti-ethofenprox antibody.
7. The pesticide ethofenprox fluorescence quantitative immunochromatography detection test paper card comprises the test paper strip of any one of claims 1 to 6 and a cover plate which is buckled with the test paper strip and meets the following conditions: the cover plate is provided with a sample adding window corresponding to the sample pad and a display window corresponding to the detection line and the quality control line.
8. Any one of the following:
(A1) the phenylacetic acid-ethofenprox azo compound according to any one of claims 2 to 6;
(A2) a conjugate of etofenprox azoate as claimed in any one of claims 2 to 6 with carrier protein 1;
(A3) an anti-ethofenprox antibody as set forth in any one of claims 2 to 6;
(A4) an anti-ethofenprox antibody labeled with a fluorescent microsphere as set forth in any one of claims 2 to 6;
(A5) a composition consisting of a conjugate of 4-oxo-4- (3-phenoxybenzyloxy) butyric acid as claimed in any one of claims 2 to 6 and carrier protein 2, and (a 4).
9. Any of the following applications:
(B1) use of the test strip of any one of claims 1 to 6 or the test card of claim 7 or the substance of claim 8 for the detection of ethofenprox;
(B2) use of the substance of claim 8 in the preparation of the test strip of any one of claims 1 to 6 or the test card of claim 7.
10. A method for detecting the content of ethofenprox in a sample to be detected comprises the following steps:
(C1) drawing a standard curve: preparing standard solution of etofenprox with a series of concentrations, detecting by using the test strip of any one of claims 1 to 6 or the test paper card of claim 7 respectively, adding the standard solution to the sample pad of the test strip or the sample adding window of the test paper card, and reacting for 10 min; reading the fluorescence intensity of the quality control line and the detection line by using a fluorescence reader; drawing a standard curve graph by taking the concentration of the standard substance of the ethofenprox as an abscissa and the fluorescence intensity ratio of the detection line and the quality control line as an ordinate to obtain a standard curve equation;
(C2) detecting a sample to be detected: adding the sample to be detected into the sample pad of the test strip or the sample adding window of the test strip card, and reacting for 10 min; and (3) reading the fluorescence intensities of the quality control line and the detection line of the test strip by using a fluorescence reading instrument, substituting the fluorescence intensity ratio of the detection line and the quality control line into the standard curve equation obtained in the step (C1), and calculating to obtain the concentration value of ethofenprox in the sample to be detected.
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